The ultimate goal of these studies is to determine what role, if any, deficiencies of intestinal peptide hydrolases play in human diseases. Both brush border and cytoplasmic peptide hydrolyzing enzymes will be isolated and characterized, their mechanism(s) of action will be studied, and the details of digestion and absorption of peptides from the intestinal lumen will be further investigated. Under the current project, 3 rat and 2 human brush border peptides hydrolases with different substrate specificities were isolated and a general method for determining both brush border and cytoplasmic activity in human peroral intestinal biopsy specimens was developed confirming the feasibility of this approach. To isolate cytoplasmic and additional brush border enzymes from both rat and human mucosa use will be made of acrylamide and starch gel electrophoresis and ion-exchange, affinity, and gel filtration chromatography. Substrate specificities and other biochemical characteristics will be determined for all isolated enzymes. The mechanisms of absorption of peptides by rat intestine will be studied by use of a recently developed flow-through system which minimizes the effect of cytoplasmic peptide hydrolases released by intestinal segments in vitro. The possible role of brush border gamma-glutamyl transpeptidase enzyme(s) in peptide transport and digestion will be investigated. Peroral intestinal biopsy specimens from patients with nontropical sprue, regional enteritis, ulcerative colitis, diarrhea of undetermined etiology, and idiopathic failure to thrive will be assayed for both brush border and cytoplasmic peptide hydrolase levels and compared to results from controls. Substrates used for these human studies will be dictated by specificities determined from studies of isolated human enzymes.